Biomedical subjects

V Lafont

Publications and source records attributed to V Lafont.

At least 19 recordsLinked to original sources

PubMed2001

Isopentenyl pyrophosphate, a mycobacterial non-peptidic antigen, triggers delayed and highly sustained signaling in human gamma delta T lymphocytes without inducing eown-modulation of T cell antigen receptor.

The Vgamma9Vdelta2 T cell subset, which represents up to 90% of the circulating gammadelta T cells in humans, was shown to be activated, via the T cell receptor (TcR), by non-peptidic phosphorylated small organic molecules. These phosphoantigens, which are not presented by professional antigen-presenting cells, induce production of high amounts of interferon-gamma and tumor necrosis factor (TNF-alpha). To date, the specific signals triggered by these antigens have not been characterized. Here we analyze proximal and later intracellular signals triggered by isopentenyl pyrophosphate (IPP), a mycobacterial antigen that specifically stimulates Vgamma9Vdelta2 T cells, and compare these to signals induced by the non-physiological model using an anti-CD3 antibody. During antigenic stimulation we noticed that, except for the proximal p56(lck) signal, which is triggered early, the signals appear to be delayed and highly sustained. This delay, which likely accounts for the delay observed in TNF-alpha production, is discussed in terms of the ability of the antigen to cross-link and recruit transducing molecules mostly anchored to lipid rafts. Moreover, we demonstrate that, in contrast to anti-CD3 antibody, IPP does not induce down-modulation of the TcR.CD3 complex, which likely results in the highly sustained signaling and release of high levels of TNF-alpha.

Antigens, Bacterial↗

PubMed2000

Covariance analysis of protein families: the case of the variable domains of antibodies.

A nonrestrictive method for identifying covariance in protein families is described and applied to human and mouse germline Vkappa and VH sequence alignments. Amino acids that occur at each position in a sequence alignment are divided into two sets, called a word, by generating all possible combinations of alternative amino acids. Each word is associated with a pattern of changes. Words with identical patterns identify covariant positions. In antibody variable domains, the number of words generated ranged between 1103 and 2195 depending on the alignment, of which 4 to 12 % occurred in covariant pairs. Despite the nonrestrictive character of pattern generation, covariant residues did not reflect a random selection with respect to the nature of amino acid changes and/or their spatial proximity in a reference crystallographic structure. This approach allowed the identification of a covariance signal for positions with high variability, mostly located in the outer part of the common structural framework of antibody variable domains. Covariance in these regions may reflect the existence of alternative and mutually exclusive atomic arrangements that are compatible with antibody function. The method may be of general applicability to rationalize residue variability in protein families.

Algorithms↗

PubMed2000

The T cell antigen receptor activates phosphatidylinositol 3-kinase-regulated serine kinases protein kinase B and ribosomal S6 kinase 1.

The present study has explored T cell antigen receptor-regulated serine kinases in human T cells. The results identify two phosphatidylinositol 3-kinase (PI3K)-controlled serine kinases operating downstream of the T cell receptor (TCR) in primary T cells: (i) protein kinase B whose activation regulates the phosphorylation of glycogen synthase kinase 3 and (ii) ribosomal S6 kinase 1, a kinase with a critical role in the regulation of protein synthesis and cell growth. T cells express two isoforms of S6k1: a 70 kDa cytoplasmic kinase and an 85 kDa isoform that has a classic nuclear localisation. TCR ligation triggers a parallel engagement of both the 70 and 85 kDa isoforms of S6k1 in a response that requires PI3K function.

Antibodies↗

PubMed2000

Tumor necrosis factor-alpha production is differently regulated in gamma delta and alpha beta human T lymphocytes.

Tumor necrosis factor-alpha (TNF-alpha) plays a crucial role in the early defense against pathogens. This cytokine is produced by several cell types including T lymphocytes expressing the alphabeta as well as the gammadelta T cell receptor (TcR). In human, the circulating gammadelta T cells, which mostly express Vgamma9Vdelta2 TcR, have been strongly suggested to play an important protective role against infectious agents. These activated cells early produce high amounts of TNF-alpha, which induce a determinant beneficial effect against development of intracellular pathogens; however, sustained production of this cytokine can result in immunopathological diseases. The signals that regulate TNF-alpha production in Vgamma9Vdelta2 T cells are totally unknown. In primary alphabeta T cells, TNF-alpha production was shown to necessitate engagement of the TcR and CD28, and to be independent of the p38 mitogen activated protein kinase pathway. We demonstrate herein that, in contrast to alphabeta T cells, TNF-alpha production in Vgamma9Vdelta2 T lymphocytes is independent of CD28 costimulation and highly dependent on TcR-induced p38 kinase and extracellular signal-regulated kinase 2 pathway activation for optimal cytokine release. Moreover, we bring elements supporting the idea that the "activation threshold" of gammadelta T cells leading to cytokine production is lower than that of alphabeta T cells.

CD28 Antigens↗

PubMed2000

Antigen receptor signal transduction: activating and inhibitory antigen receptors regulate STAT1 serine phosphorylation.

Antigen receptors are crucial regulators of the mammalian immune response. Immediate antigen receptor proximal signal transduction pathways mediated by tyrosine (Tyr) kinases are well defined. In contrast, much less is known about the network of serine (Ser) kinases and Ser kinase substrates that are linked to antigen receptor function. Here we describe a new signaling module for antigen receptors in lymphocytes; a Ser kinase pathway that phosphorylates Ser 727 in STAT1alpha, a member of the signal transducer and activator of transcription gene family. In the present study we have explored the regulation of STAT1 Ser 727 phosphorylation in human T and B lymphocytes and show that it is controlled by both positive and negative antigen receptor signaling cascades. Ligation of antigen receptors in both B and T cells induce a delayed but then sustained phosphorylation of STAT1 on Ser 727. STAT1 Ser phosphorylation is induced by the TCR in the absence of STAT1 Tyr phosphorylation, indicating that in T cells STAT1 Ser and Tyr phosphorylation are independent events. Antigen receptor regulation of STAT Ser phosphorylation is dependent on phosphatidylinositol 3-kinase-mediated signals. Furthermore, the negative regulatory receptor FcgammaRIIb, which mediates vital feedback control of B cell responses, prevents antigen receptor-induced phosphorylation of STAT1 Ser 727. The ability of antigen receptors to both positively and negatively regulate STAT1 Ser 727 phosphorylation reveals a Ser kinase network that operated during sustained responses to antigen receptor engagement.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2000

Evaluation of the supervisory system in elderly subjects with and without disinhibition.

UNLABELLED: Disinhibition and irritability, defined as loss of behavioral and emotional control, are frequent in the elderly. The working hypothesis for this study was that these disorders are associated with a cognitive alteration of control processes that manifests as non-routine behavior because of the dysfunction of a general executive component known as the supervisory attentional system (SAS). METHODS: A total of 28 elderly subjects with mild cognitive impairment were recruited and divided into two groups using the Neuropsychiatric Inventory. Fourteen subjects were allocated to the disinhibited group and 14 subjects matched for age, sex and educational level formed a disinhibition-free control group. The neuropsychological battery included the following tests: Mini Mental Score Evaluation, Boston Naming test, Token test, Trail Making and Verbal Fluency. Two tasks were specifically designed to stress the SAS: 1) A specific verbal sentence arrangement task in which subjects had to use sequential reasoning with verbal material. Each test sequence consisted of a series of words shown in jumbled order. The construction of some sequences had to be done by using familiar routine associations (valid conditions). In contrast, other sequences required the overriding selection of familiar routine associations, which were inappropriate within the general context of the task (invalid conditions). 2) Using the Continuous Performance Test, four aspects were evaluated: sustained, selective, preparation and suppressive attention. RESULTS: The only group differences in neuropsychological test results were the following: 1) the sentence arrangement task. In comparison with the control group, the disinhibited group was impaired in invalid conditions and the calculated difference between the number of correct responses in invalid conditions minus that in valid conditions was significantly higher; and 2) the CPT. Disinhibited subjects had a significantly lower number of hits, exclusively in the 'suppressive attention' paradigm. These results suggest that subjects with disinhibition have impaired supervisory system function.

Aged↗

PubMed1999

Evidence for a p21(ras)/Raf-1/MEK-1/ERK-2-independent pathway in stimulation of IL-2 gene transcription in human primary T lymphocytes.

T cell stimulation leads to triggering of signals transmitted from the cell membrane to the nucleus through TCR/CD3 proteins. Characterization of these signals largely results from the use of cell lines stimulated with anti-CD3 monoclonal antibodies. These studies have established that activation caused a rapid increase in the formation of GTP-bound Ras, which stimulates the mitogen-activated protein kinase pathway involving the extracellular-regulated kinase-2 (ERK-2) and activates the nuclear factor of activated T cells (NF-AT) that regulates interleukin-2 (IL-2) gene transcription. In the present study, we used human primary T cells, and we investigated the intracellular signals triggered by two different anti-CD3 monoclonal antibodies (UCHT1 and X-35), which both strongly induce cell proliferation. We found that, in contrast to the commonly used UCHT1, X-35 activated IL-2 gene transcription without stimulation of the Raf-1/mitogen-activated ERK kinase-1 (MEK-1)/ERK-2 phosphorylation cascade; we also showed that X-35 stimulation, which triggers an ERK-2-independent pathway, does not involve activation of p21(ras). In addition to demonstrating that activation of p21(ras) and of its Raf-1/MEK-1/ERK-2 effector pathway is not an event obligatorily triggered upon TCR/CD3 ligation, these results provide the first evidence of the existence of a p21(ras)/ERK-2-independent pathway for IL-2 gene transcription in human primary T lymphocytes.

Cells, Cultured↗

PubMed1998

Effector pathways regulating T cell activation.

Activation of T lymphocytes is a key event for an efficient response of the immune system. It requires the involvement of the T cell receptor antigen as well as costimulatory molecules such as CD28. Engagement of these receptors through the interaction with a foreign antigen associated with major histocompatibility complex molecules and CD28 counter-receptors B7.1/B7.2, respectively, results in a series of signaling cascades acting in synergy and which culminate in activation of interleukin-2 gene transcription and eventually cell proliferation. Many studies aimed at characterizing these specific effector pathways have been published; however, the actual signaling molecules that transduce activation signals from the cell membrane to the nucleus and that directly regulate interleukin-2 gene transcription are not yet completely defined and remain a matter of debate. In this commentary, we have attempted to analyze the results, which are sometimes diverging if not totally contradictory, characterizing effector pathways that possibly are triggered during T cell activation.

Animals↗

PubMed1998

Initiation and supervisory processes in schizophrenia and depression.

Clinical studies show that schizophrenic and depressive subjects have problems with daily life activities, and neuropsychological studies tend to explain these problems in terms of a dysexecutive syndrome. Verbal fluency and sentence arrangement are tasks considered to focus on two aspects of the dysexecutive syndrome known as initiation and supervision processes, respectively. In this study, we assessed performance in these two tasks in schizophrenia and depression. Twenty-six schizophrenic subjects (chronic schizophrenia, DSM IV definition) were compared with 26 control subjects balanced for sex, age and educational level, and 16 depressive subjects (major depression episode, DSM IV) were compared with 11 similarly balanced control subjects. Switching and clustering scores were evaluated during a semantic fluency task as two components underlying the initiation and organization processes. Capture errors specific to failure of the supervisory system and differences between the number of correct responses in two conditions (valid/invalid) were evaluated as indexes of the supervision process in a sentence arrangement task. In the semantic fluency task, switching scores were significantly lower in the schizophrenic and depressive subjects than in their respective controls. In the sentence arrangement task, only the schizophrenic subjects made significantly more capture errors than their controls and had significantly fewer correct sequences in invalid conditions than in valid conditions. This study shows a dissociation between supervision and initiation processes in two different psychiatric populations. Initiation is impaired, but supervision is preserved in depression, whereas both initiation and supervision are impaired in schizophrenia.

Adult↗

PubMed1998

The Raf-1/mitogen-activated protein kinase kinase-1/extracellular signal-regulated-2 signaling pathway as prerequisite for interleukin-2 gene transcription in lectin-stimulated human primary T lymphocytes.

It has been shown that stimulation of lymphoid cells causes the activation of the extracellular signal-regulated-2 (ERK-2) which activates nuclear factor of activated T cells (NF-AT), a transcription factor involved in the regulation of interleukin-2 (1L2) gene transcription. ERK-2 is activated via a kinase cascade initiated by activation of the G protein p21Ras followed by phosphorylation and activation of Raf-1 and mitogen-activated protein kinase kinase-1 (MEK-1). Activation of this pathway has been described primarily in human T cell lines; however, using primary T lymphocytes from transgenic mice, a recent study has shown that a blockade of this cascade did not perturb lymphocyte stimulation and proliferation. In the present paper, we studied in human primary T cells the possible involvement of the Raf-1/MEK-1/ERK-2 pathway upon stimulation by jacalin, a mitogenic lectin which specifically stimulates CD4+ lymphocytes. We show here that the mitogen-activated protein (MAP) kinase pathway was stimulated in human purified lymphocytes upon activation with jacalin. Moreover, activation of this pathway appeared to be essential, since its blockade by a specific inhibitor of the MEK-1 kinase abolished IL2 gene transcription; in contrast, in T cells stimulated with phytohemagglutinin M(PHA), another potent T cell mitogenic lectin, blockade of MEK-1 reduced but did not totally inhibit either ERK-2 phosphorylation or IL2 mRNA expression. This shows, as already suggested, that another pathway in addition to the Raf-1/MEK-1/ERK-2 kinase cascade could be triggered in T cell activation. Jacalin stimulation therefore appeared to be a good model for the specific activation of the MAP kinase pathway in human primary T lymphocytes, which would allow the characterisation of drugs specifically targeted to this particular pathway.

Gene Expression Regulation↗

PubMed1998

Clustering and switching strategies in verbal fluency tasks: comparison between schizophrenics and healthy adults.

Verbal fluency tasks are frequently used in clinical neuropsychology. Clustering (the production of words within semantic subcategories) and switching (the ability to shift between clusters) have been described as 2 components underlying fluency performance. We compared the use of clustering and switching in schizophrenic patients and healthy subjects. Seventy-eight schizophrenic subjects (DSM-IV criteria) and 64 control participants matched for age and educational level were recruited. Negative, disorganized, and productive clinical dimensions were evaluated using the SANS and SAPS scales. The number of words generated per semantic-phonemic cluster and the number of switches were evaluated during 2 verbal fluency tasks (phonemic and semantic). In the healthy controls switching and clustering were closely related to the total number of words generated in the verbal fluency tests. The role of the 2 components was partly dependent on the specific task. Switching was prevalent in formal fluency, while both switching and clustering contributed to semantic fluency. In comparison to the healthy controls, the overall group of schizophrenic patients showed a significant impairment of switching in the formal fluency task and of both switching and clustering in the semantic fluency task, and both the negative and disorganized dimensions correlated with verbal fluency performance, the number of swtiches during the phonemic fluency task, and the clustering during semantic fluency task.

Adult↗

PubMed1997

Transferrin receptor functions as a signal-transduction molecule for its own recycling via increases in the internal Ca2+ concentration.

Transferrin binding to its receptor modulates transferrin receptor (Tf-R) recycling rates in several cells [Klausner, R. D., Van Renswoude, J., Ashwell, G., Kempf, C., Schechter, A., Dean, A. & Bridges, K. R. (1983a) J. Biol. Chem. 258, 4715-4724; Gironès, N. & Davis, R. J. (1989) Biochem. J. 264, 35-46; Sainte-Marie, J., Vidal, M., Bette-Bobillo, P., Philippot, J. R. & Bienvenüe, A. (1991) Eur. J. Biochem. 201, 295-302]. To delineate the mechanism of this regulation, we hypothesized that the binding of the ligand to its receptor could lead to activation of several second-messenger pathways, which may redundantly stimulate recycling of the receptor. The effects of different regulators of Ca2+ flux or concentrations were investigated on the Tf-R-recycling pathway; these studies were carried out in two cell types. Perhexiline, a calcium antagonist, slowed receptor recycling in comparison with the control by more than 80% in L2C cells and by 60% in Jurkat cells (B and T lymphoblasts, respectively) but did not affect their internalization rate. Perhexiline thus trapped considerable amounts of Tf-R in the internal compartment. Ca2+ chelators, such as EGTA or 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid, and a Ca2+-channel inhibitor (Ni2+) decreased drastically the recycling rate of Tf-R. Tf-R recycling was shown to be slowed by a calmodulin antagonist. Conversely, artificial elevation of free internal Ca2+ in L2C cells, using lectin, accelerated the recycling rate. These results suggest that the intracellular Ca2+ concentration plays an important role in the outward flow of transferrin receptors. Consequently, we examined the role of transferrin in internal free Ca2+ regulation. The addition of transferrin or anti-(Tf-R) Ig specifically elicited a rise in [Ca2+], as demonstrated by inefficacy of apotransferrin or irrelevant antibodies. These results suggest that Ca2+ is a regulator of Tf-R recycling and that Tf-R seems to function as a signal-transduction molecule (perhaps in conjunction with other membrane proteins) rather than merely as an endocytic receptor.

Antibodies↗

PubMed1997

The lectin jacalin specifically triggers cell signaling in CD4+ T lymphocytes.

The lectin jacalin was shown to specifically stimulate CD4(+) lymphocytes. This lectin, which presents a peptide highly similar to a sequence of the HIV external glycoprotein, interacts with CD4 and is able to inhibit in vitro HIV infection. Since jacalin binds also CD8, its mitogenic specificity cannot exclusively be attributed to its interaction with CD4. We therefore hypothesized that the lectin could trigger signals specifically associated with CD4. Here we show that jacalin triggers IL2 gene transcription only in CD4(+) lymphocytes. In parallel, we show that numerous proteins are tyrosine phosphorylated in this cell subset while only a restricted number of them are phosphorylated in CD8(+) cells. Moreover, we show that the tyrosine kinase p56lck, which is associated with both CD4 and CD8, is activated only in CD4(+) lymphocytes, making this lectin a good model for the study of cell signaling triggered in this restricted subpopulation.

CD4 Antigens↗

PubMed1997

Evidence for a CD4-associated calcium influx independent of the phosphoinositide transduction pathway in human T cells.

We recently showed, using human Jurkat T cell variants lacking the T cell receptor (TCR)/CD3 complex, that the lectin jacalin is able to trigger intracellular calcium increase provided that CD4 is expressed on the cell surface. Involvement of the CD4 molecule in jacalin-induced biological effects was furthermore demonstrated in differentiated U937 myelomonocytic cells expressing or not expressing CD4, and is confirmed here in human CD4-transfected mouse thymoma cells. In the present paper, we analyze the CD4-associated calcium response triggered by jacalin independently of the TCR/CD3 complex. We show that the observed calcium rise results from a direct long-lasting calcium influx from the outside without release of calcium from intracellular stores. We demonstrate that it is independent of the phosphoinositide phospholipase C transduction pathway. Moreover, we show that this peculiar calcium response can be blocked by protein tyrosine kinase inhibitors (herbimycin and genistein) giving evidence of the involvement of a protein tyrosine kinase, the best candidate of which is the CD4-associated p56lck. Altogether, our results suggest that, independently of the TCR/CD3 complex, CD4 may be involved in the triggering of a calcium signal dependent on a protein tyrosine kinase and independent of the phosphoinositide transduction pathway.

Animals↗

PubMed1997

Interaction of Yersinia enterocolitica with macrophages leads to macrophage cell death through apoptosis.

Suppression of the host defense is one of the hallmarks of Yersinia enterocolitica infection. This enteric pathogen resists phagocytosis and interferes with macrophage functions from an extracellular localization (oxidative-burst generation and tumor necrosis factor alpha production). In this study, we investigated the fate of the Y. enterocolitica-infected macrophage. We found that murine J774A.1 macrophages and macrophages derived from human monocytes were killed by infection with Y. enterocolitica. Analysis of cellular morphology and DNA fragmentation revealed that macrophage cell death occurs through the induction of apoptosis. A total of 92% +/- 5% (mean +/- standard deviation) of murine J774A.1 macrophages and 74% +/- 6% of human monocyte-derived macrophages underwent apoptosis upon Yersinia infection after 4 and 20 h, respectively. The broad-spectrum caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone blocked completion of the Yersinia-induced apoptotic program but not the surface exposure of phosphatidylserine as an early-stage apoptotic event. Analysis of different Yersinia mutants showed that macrophage apoptosis depends on a functional Y. enterocolitica type III protein secretion system. Apoptotic cell death of macrophages was not related to the YopE-mediated cytotoxic effect of Yersinia, since disruption of actin microfilaments by a Y. enterocolitica strain expressing a restricted repertoire of yop genes, including YopE, did not result in macrophage apoptosis. Furthermore, Yersinia-induced cytotoxic alterations in epithelial HeLa cells, which are conferred by YopE, did not lead to apoptosis. Our data demonstrate for the first time that Y. enterocolitica promotes the apoptosis of macrophages, an effect which is clearly distinct from the morphological alterations mediated by Yersinia on epithelial HeLa cells.

Actins↗

PubMed1996

Differential effects of macrophage- and granulocyte-macrophage colony-stimulating factors on cytokine gene expression during rat alveolar macrophage differentiation into multinucleated giant cells (MGC): role for IL-6 in type 2 MGC formation.

Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage (GM)-CSF stimulate the differentiation of rat alveolar macrophages (AM) into multinucleated giant cells (MGC) with distinct phenotypes (type 1 and type 2 MGC). In the present study, we analyzed the profile of cytokine gene expression induced respectively, by M-CSF and GM-CSF during rat AM differentiation using reverse transcription-PCR. Enhanced mRNA expression for IL-1alpha, TNF-alpha, and TGF-beta1 was observed 3 h after treatment with M-CSF (50 U/ml) or GM-CSF (50 U/ml). In contrast, IL-6 mRNA expression was increased by GM-CSF but not M-CSF. Kinetic analysis indicated that GM-CSF stimulated IL-6 expression early (1.5 h), with maximal effect observed at 24 h and up to 5 days thereafter. Increased mRNA levels for IL-6 were associated with higher IL-6 activity in the culture media of differentiating AM. IL-6 activity was stimulated 3 h after treatment with GM-CSF and increased with time (up to 5 days). Interestingly, addition of exogenous IL-6 (20-100 ng/ml) alone or in combination with GM-CSF to AM cultures increased slightly and selectively the formation of MGC with type 2 phenotype. Conversely, neutralization of endogenous IL-6 during AM differentiation into MGC inhibited significantly (up to 50%) the formation of type 2 MGC. These results suggest a role for IL-6 in the formation of type 2 MGC and provide some insights into the mechanisms of MGC formation and the processes that regulate it positively.

Animals↗

PubMed1996

The lectin jacalin triggers CD4-mediated lymphocyte signaling by binding CD4 through a protein-protein interaction.

The lectin jacalin specifically stimulates lymphocytes expressing the CD4 antigen. Recent studies have also demonstrated that this lectin interacts with CD4, inhibits in vitro HIV infection, and triggers cell signaling directly via CD4. Jacalin as a lectin was suggested to trigger CD4-mediated cell signaling by interacting with the oligosaccharide side chains of CD4 located on Asn271 and Asn3OO. Such a hypothesis was of importance because it implied that the glycosylated chains could represent a functional domain directly involved in CD4-related cell activation. We analyzed this possibility by studying the effect of hapten sugars on jacalin-induced CD4 cell signaling and jacalin/CD4 interaction, and by studying the binding capacities of the lectin toward glycosylated, deglycosylated, and unglycosylated CD4. The results presented in this study provide evidence that jacalin does not recognize the CD4 oligosaccharide chains and actually binds CD4 through a specific protein-protein interaction; as a consequence these results rule out the involvement of the CD4 saccharide moieties in CD4-mediated cell signaling triggered by the lectin.

Base Sequence↗

PubMed1994

Jacalin, a lectin that inhibits in vitro HIV-1 infection, induces intracellular calcium increase via CD4 in cells lacking the CD3/TcR complex.

The lectin jacalin interacts with the CD4 cell surface antigen; this lectin inhibits in vitro infection by human immunodeficiency virus type 1 without preventing virus binding on the host cell. The infection process is known to involve cellular events triggered by the binding of the viral external glycoprotein gp120 to CD4. Herein we demonstrate that jacalin induces cell signaling directly through the CD4 antigen and that independently of the CD3/TcR complex. The capacity of jacalin to trigger cell signals through the CD4 molecule is discussed in relation to its ability to inhibit HIV infection.

CD3 Complex↗